Phytochemical profiling, antimicrobial, and antioxidant activities of hydroethanolic extracts of prickly pear (Opuntia ficus indica) fruit and pulp

Abstract Phenolic compounds in prickly pear [Opuntia ficus indica (L.) Mill.] are known to contribute to the antioxidant and antimicrobial activities of the prickly pear. The present study aimed to evaluate the antioxidants and in vitro antimicrobial potential in the hydroethanolic extracts of different parts (fruit, cladode, and pulp) of prickly pear. Different polyphenolic compounds were analyzed by using high‐performance liquid chromatography. The results indicated that cladode possessed a higher quantity of phenolics compared with that observed in fruit and pulp. The most important phenolic compound in high quantity was gallic acid (66.19 μg/g) in cladode. The 100% aqueous extract of cladode exhibited the highest antioxidant (92%) and antimicrobial activities against Salmonella typhi (3.40 mg/ml), Helicobacter pylori (1.37 mg/ml), Escherichia coli (1.41 mg/ml), and Staphylococcus aureus (1.41 mg/ml). Principal component analysis (PCA) indicated that antioxidant activity and minimum inhibitory concentration (MIC) responses had a significant negative correlation with each other. Overall, the current results provided basic data for choosing prickly pear cladode with high antioxidant capacity for the development and consumption of antioxidant‐based alternative medicines and value addition of formulated foods.


| INTRODUC TI ON
The increasing demands of water and feed resources across the world's dry areas require alternative sources of animal feed, specifically crops with better water-use efficiency (Ameer, Bae, Jo, Lee, et al., 2017). One alternative with the potential for widespread contribution toward reducing the impact of reduced feed and water availability is the cactus pear. Cactaceae Juss (1789) is a highly diversified family of xerophytes that are dominant in the arid and semi-arid environments of the Americas, which is its center of origin and diversification (Hernández-Hernández et al., 2014). Cacti comprise approximately 1400-1800 described species in the world (Guerrero et al., 2019), and Mexico is the country with the greatest diversity, with 52 genera and 850 species, of which an estimated 84% are endemic (Guerrero et al., 2019). Nearly, 31% of cacti are globally threatened (Goettsch et al., 2015) due to changes in land use, the introduction of exotic species, and uncontrolled harvesting of these plants for use as food, raw materials, and other purposes as well as influences by climate change. Some of them are listed by the International Union for the Conservation of Nature (IUCN) under various threat categories which necessitate conservation efforts. Opuntioideae is the richest genus within the Cactaceae of nearly 200 described species (Huo et al., 2022;Majure et al., 2012;Majure & Puente, 2014). Opuntia species are highly distributed throughout arid and semi-arid environments and are well-adapted to drought-stressed conditions (Aliscioni et al., 2021;Majure et al., 2012). The genus Opuntia is endemic to the Americas and distributed from regions of Canada to Argentina (Majure & Puente, 2014), showing a high number of regional endemic species in Mexico (González-Elizondo et al., 2017). Prickly pear fruit has an aqueous pulp and contains 87.5% of water. It has a low energy value of 170 kJ/100 g. Its carbohydrate content, mainly glucose and fructose, provides 94% of the energy value. It exhibits a low titratable acidity of 1.83 g citric acid/kg than oranges, pineapple, and bananas (Garcia-Amezquita et al., 2018).
In recent years, much attention has been devoted to natural antioxidants and their association with health benefits provision (Ameer, Chun, et al., 2017). Plants are potential sources of natural antioxidants.
It produces various antioxidant compounds to counteract reactive oxygen species (ROS) in order to survive (Jiang et al., 2021). ROS, which include free radicals, such as superoxide anion radicals (O −2 ), hydroxyl radicals (-OH), and nonfree radical species, like H 2 O 2 and single oxygen are various forms of activated oxygen Ameer, Bae, Jo, Chung, et al., 2017;Kang et al., 2022). These molecules are exacerbating factors in cellular injury and aging processes. In foods, ROS can cause lipid peroxidation, which leads to the deterioration of the foodstuffs (Li et al., 2020;Yang et al., 2021). The oxidative deterioration of lipid-containing foods is responsible for the rancid odors and flavors during processing and storage, consequently decreasing the nutritional quality and safety of foods due to the formation of potentially toxic secondary compounds. The addition of antioxidants is a method for increasing the shelf life of foodstuffs Jiang, Lee, Ameer, & Eun, 2019;Lee et al., 2002). The antioxidant activity of prickly pear is comparable to that of red oranges and grapes (Cano et al., 2017). It exerts biological effects, which may be due to the synergistic action of betalains (tyrosine-derived pigments), flavonoids, and other biologically active components (Stintzing et al., 2005).
All parts of the cactus plant are rich in polyphenols including various flavonoids and phenolic acids. The total phenolic content (TPC) of prickly pear fruit pulp has been reported up to 218.8 mg gallic acid equivalents (mg GAE)/100 g fresh fruit in red-skinned fruit. Prickly pear fruit is considered a rich source of flavonols. The quantification of five types of flavonoids showed that quercetin was the predominant one (58.7% ± 54.3%), followed by isorhamnetin (31.7% ± 18.8%), luteolin (11.5% ± 5.4%), and kaempferol (11.0% ± 4.8%; Fernández-López et al., 2010). The analysis of the peels showed that isorhamnetin glycosides, especially isorhamnetin-3-O-rutinoside, are the main flavonol glycosides in prickly pear peels (Moussa-Ayoub et al., 2011). More than 20 polyphenolic compounds, including flavonoids and tannins were also detected in the prickly pear seeds.
Cactus pear contains bioactive compounds, such as quercetin, isorhamnetin, and kaempferol. These bioactive compounds have significant antimicrobial activity against different microbes including Bacillus subtilis, Escherichia coli, Psedomonas auregnosa, and Klebsiella pneumonia (Nenaah, 2013). Food-borne bacteria resulting in gastroenteritis in humans is Campylobacter. The aqueous, ethanolic, and methanolic extracts of Opuntica ficus indica have significant antimicrobial activity against Vibrio cholera (Sánchez et al., 2010). Prickly pear (O. ficus indica) has pharmacological significance because it prevents the disorders of gut and diseases like food poisoning due to Vibrio cholera and Campylobacter. Belay et al. (2015)

| Raw material collection and preparation
Red pink-colored prickly pear fruits of the cultivar (Meyer) with cladodes were brought from Talagang district, Chakwal, Pakistan.
The samples were carried to the laboratory of the Institute of Food Science and Nutrition, University of Sargodha, Sargodha. Fruits and cladodes were washed separately to remove dirty materials, airdried, and stored in air-tight glass containers at 4-6°C for use in different analyses.

| Preparation of aqueous and ethanolic extracts of cladodes
Fresh cladodes extraction was carried out according to Benayad et al. (2014) with few modifications. Cladodes (20 g) were ground and mixed with different concentrations of solvents (ethanol and water) as shown in Table 1, which were then put in a separate conical flask and these were placed in a shaking incubator (Shing Saeng Skir-601L) at 22-25°C for 24 h. Macerated extracts were then filtered using Whatman No. 1 filter paper. Then, water and ethanol were evaporated in a rotary evaporator (Heidolph Laboratory,4001) at 45°C and 60 rpm (Benayad et al., 2014).

| Total phenolic content
TPC was determined following the protocol described by AOAC method number 2017.13. For the assay, 0.25 ml of sample was taken in test tubes, mixed with 1.25 ml 10% Folin-Ciocalteu reagent, and diluted with distilled water 10 times. This solution was mixed with 1 ml of 7.5% sodium carbonate and incubated for 30 min in the dark.
The absorbance was measured at 765 nm by spectrophotometer (Shimadzu Instruments). The TPC was expressed as gallic acid equivalents in milligrams (mg GAE mg)/100 g of FW.
2.4.2 | Total flavonoid content TFC was determined following the method of Chougui et al. (2013) with few modifications. For the reaction, 1.5 ml of extract was taken in a test tube and incubated with 2% of 1.5 ml AlCl 3 reagent for 30 min in darkness. The absorbance was recorded at 430 nm by the spectrophotometer. A calibration curve was prepared using quercetin as the standard. The results were expressed as mg equivalent of quercetin (mg QE)/100 g of FW (Chougui et al., 2013).

| Total carotenoids content
Chlorophyll contents (Chl a and Chl b) were determined by spectrophotometry through the reported method of Braniša et al. (2021).
Total carotenoid content was quantified with few modifications.
Ethanol-water in the ratio of 4:1 was employed as the extraction solvent. For the assay, 5 ml of sample was centrifuged for 5 min at 5000 rpm at 5°C in a Z383K Hermle centrifuge. The top layer of solvent had been recovered and transferred to a 25 ml volumetric flask.
The spectrophotometric absorption spectra were recorded for Chl a, Chl b, and total carotenoid content at wavelengths of 663.6, 647, and 470 nm, respectively. Absorbance was measured at 450 nm by the spectrophotometer (Shimadzu Instruments). The results were expressed as the microgram equivalent of beta carotene (μg/ml).
Following equations were used for the measurement of Chl a, Chl b, and total carotenoid contents.

| Free radical scavenging activity by DPPH
Free radical scavenging activity was determined by using the method of Shen et al. (2010) with minor modifications according to AOAC SMPR method number 2011.011. For the assay, 750 μl of the extract was added to 1.75 ml of 0.02 g/L DPPH dissolved in ethanol. This mixture was incubated for 30 min in the darkness. The absorbance was measured at 517 nm against control by the spectrophotometer.
Ascorbic acid was taken as the reference compound. The free radical scavenging activity of DPPH has been calculated by using the following formula: (1) Chlorophyll a g ml = 12.25 A 663.6 -2.25 A 646.6 , where A 0 is the absorbance of the control and A 1 is the absorbance of the extract.

| High-performance liquid chromatography
Flavonoids and phenolics were quantitatively analyzed by HPLC as 0.05 μL) were prepared. From 1 to 11, columns were labeled with these dilutions. The sample was poured into columns from 1 to 11. Bacterial inoculum was prepared to the size of 10 4 -10 5 CFU/ mL. 5 μL of bacteria was poured into wells in columns 1-12. Plates were incubated at 37°C for 12-18 h. Growth was checked by naked eye evaluation. was considered to be statistically significant.

| Total phenolic content
The results showed significant variation (p < .05) in the TPC of cactus pear's cladode, pulp, and fruit between parts and solvent fractions ( Figure 1a;

| Total flavonoid content
The results of TFC regarding the influence of parts of Opuntia cladode, pulp, and fruit are presented in Figure 1b and Table 1. It was evident from the results that TFC of cactus pear cladode, pulp, and fruit found to be significantly (p < .05) different among different parts of the prickly pear plant. The highest TFC was observed in cactus pear cladode, whereas the lowest value was observed in pulp (p < .01).
With respect to solvent fractions, it was found that aqueous extracts yielded the highest TFC, whereas the lowest value was found in the case of ethanolic extracts (p < .01).

| Total carotenoids content
Opuntia cladode contained significantly (p < .05) the highest carotenoids, whereas the pulp exhibited the lowest carotenoids ( Figure 1c; Table 1). With respect to solvent fractions, it is also observed that the total carotenoid contents of cactus pear cladode, pulp, and whole fruit decreased significantly (p < .05) with a corresponding decrease in ethanol concentration (p < .01). Aqueous extracts exhibited the highest total carotenoid contents, whereas the lowest total carotenoid contents were found in ethanolic extracts.

| Free radical scavenging activity (DPPH)
The results of the DPPH scavenging activity are presented in Figure 1d and

| Antimicrobial activity of the extracts
MIC of extracts was higher against Staphylococcus aureus followed by Helicobacter pylori, Escherichia coli, and Salmonella typhi ( Figure 2a and

| Interaction between Opuntia parts and microbes
The results with respect to the interaction between microbes and cactus pear parts on MIC of hydroethanolic extracts of cactus pear cladode, pulp, and whole fruit are presented in Figure 2b and

| Interaction between Opuntia parts and solvent fractions
The results regarding the interaction between cactus pear parts and solvent fractions on the MIC of cactus pear cladode, pulp, and whole fruit are depicted in Figure 2c and F I G U R E 1 Determination of (a) total phenolic content, (b) total flavonoid content, (c) total carotenoids, and (d) free radical scavenging activity (DPPH) of hydroethanolic extracts of Opuntia cladode, pulp, and fruit

| HPLC of phenolic compounds in prickly pear cladode, pulp, and whole fruit
The characterization and quantification of phenolic components of prickly pear cladode, pulp, and whole fruit was carried out using HPLC. The results showed that more phenolic compounds were observed in cactus pear cladode followed by pulp and whole fruit (

| Principle component analysis
Principle component analysis (PCA) was used to determine the correlation between antioxidant activity, MIC, and the various hydroethanolic extracts. PCA bi-plot showing the relationship between antioxidant activity and microbes is presented in Figure 3. PC 1 (62.58%) and PC 2 (22.24%) explained 84.82% of the total variation (Figure 3a,b)  other vegetables and it was demonstrated that cladode had higher carotenoids than baby beetroot, carrots lettuce, and spinach. The cladodes had several times more carotenoids compared with that fruit pulp. The discrepancies in these reported carotenoid levels might be due to differences in the ripeness of the fruit because the carotenoid levels can change dramatically during ripening (Mabrouki et al., 2015;Lanuzza et al., 2017;Vuong et al., 2002). The degree of ripeness is thought to be a factor influencing the discrepancies in carotenoid quantity in Opuntia. This is consistent with the findings of Rodriguez et al. (1976) who studied carotenoids in bitter melon and reported that the number of carotenoids increased from five in the immature cladode to six at the mature-green and to 14 at the partly ripe to ripe stages (Vuong et al., 2002). Naturalistic antioxidants like phenolics, flavonoids, and carotenoids are found in different plant products (Farag et al., 1989;Jeong et al., 2004) and these are widely reported to preserve components of food which are able to oxidize easily because of oxidation. This effect differs vastly relying on the growing conditions, extraction process, and a multitude sides of the chemical structure of the active constituents, that is, the amount and position of hydroxyl groups, molecular weight, particle size, solvent concentration, time of contact, tempera- Some other researchers reported that flavonoids (quercitin) are the main compounds responsible for the scavenging activity of cladode Azizah et al., 1999;Lee et al., 2002).  Robards (2003).
Phytochemical screening revealed that several classes of secondary metabolites exist, such as flavonoids, phenolics, and carotenoids. Several molecules are active on pathogenic microorganisms (Awouafack et al., 2013;Erfan & Marouf, 2019;Tsopmo et al., 2013;Qayyum et al., 2016;Syukriah et al., 2014). The presence of these phytochemicals in the tested plant extracts can give a preliminary explanation of their antimicrobial activities. Differences were observed in the antibacterial activities of the extracts. These could be due to the differences in their chemical composition as well as in the mechanism of action of their bioactive constituents. All the extracts are rich in different phytochemicals; however, activity does not depend only on these phytochemicals in the plant extracts but also on their concentration and the possible interaction with other components (Dangoggo et al., 2012;Rodriguez-Amaya et al., 2001;Simoes et al., 2009) strains in the present study might be due to variation extraction methods, size of inoculum, incubation length, and range of solvent quantity used as reported by Moosazadeh et al. (2014) and Kalil et al. (2014).
Low antimicrobial action of extracts might be due to the variation in the type of solvent used, unreleased/bound phenols in extract matrices (Avila-Nava et al., 2014;Zeghad et al., 2019), and/or inability of extracted compounds to diffuse into the antibacterial assay medium (Kurek et al., 2011). Aqueous extracts exhibited significantly (p < .05) higher efficiency followed by ethanolic extracts against all tested pathogenic bacteria as indicated in Figure 4a. The aqueous extract activity showed that the solubility of antimicrobial potential components present in Opuntia is high in water. Mukonowenzou et al. (2021) proposed that the capacity of water to extract different antimicrobial components reported in the current research is well supported by earlier research concluding that water is a good solvent to extract antimicrobial components from medicinal plants (Mukonowenzou et al., 2021;Yu et al., 2014). The findings also revealed that Staphylococcus aureus showed more sensitivity to cactus pear fruit and pulp followed by Helicobacter pylori, Escherichia coli, and Salmonella typhi, whereas Helicobacter pylori showed more sensitivity to cladode. Staphylococcus aureus was easily inhibited at the lowest concentration in the case of fruit, whereas in the case of pulp the highest concentration of the extract was required to inhibit growth. On the other hand, a high concentration of extracts was required to inhibit the growth of Salmonella typhi. This might be due to differences in their sensitivity to antibacterial agents. The results of this study are in line with the findings of Karima et al. (2013). A similar trend was observed by Taguri et al. (2004) who reported that Gram (+) was more sensitive than Gram (−) bacteria. Staphylococcus aureus as Gram (+) bacteria is famous due to its high sensitivity to phenolic extracts. Generally, Gram (−) is more resistant to bactericidal polyphenols than Gram (+) bacteria. According to Taguri et al. (2004), the average MIC values of extracts indicated that Staphylococcus aureus (192 μg/ml) are more susceptible to polyphenols followed by Salmonella typhi (795 μg/ml) and Escherichia coli (1519 μg/ml). The present findings are in conformity with the results of Taguri et al. (2004). Ikigai et al. (1993) proposed that two factors, such as repulsion between lipopolysaccharide-coated surfaces of Gram (−) bacteria and the phenolics are responsible for this difference. It was reported in another report that gram-positive bacteria have a cell wall made up of peptidoglycan which helps in cell wall penetration (Koubaa et al., 2015). The phenolic composition of plant extracts might be responsible for their inhibitory effect against pathogenic bacteria (Rodriguez et al., 1976). The inhibitory effect of these phenolics could be explained by adsorption to cell membranes, interaction with enzymes, or deprivation of substrate and metal ions (Baydar et al., 2004).
To obtain an overall perspective of the free radical capacity and the respective chemical constituents, pairwise correlations among total phenolic content, total flavonoids content, total carotenoids, antioxidant activity, and MIC were performed. The results of the present study revealed that flavonoids, carotenoids, and DPPH were clustered together but did not seem to be associated with phenolics. On the other hand, Salmonella typhi, Helicobacter pylori, Escherichia coli, and Staphylococcus aureus are clustered together.
The interesting property of the extracts is that they have low MIC and possessed weak correlation and a lower value of MIC indicated that these extracts have high antibacterial activity against microbes. All microbes had a significant negative correlation with phenolics. These results are in concordance with the previous study conducted by Daglia (2012) who reported that phenolics have been shown to possess strong antibacterial activity. Dutta, Ghosal, Mitali & Palash (2012) also found a high correlation between total phenolics and DPPH free radical scavenging activity suggesting that phenolic compounds are the major contributors to antioxidant activity. Another study conducted by Gil et al. (2002) reported that phenolics had a significant correlation with DPPH.
The correlation of the DPPH assay with total phenolics and total flavonoids was positive, demonstrating that this assay can be considered for measuring the free radical scavenging capacity of Opuntia. It is also noteworthy that Aqu 100% of fruit are lo-

| CON CLUS ION
The present work was focused on evaluating the resultant extracts obtained from the extraction of prickly pear (Opuntia cladode) pulp and fruit as a source of natural antioxidants. In general, HPLC data showed that prickly pear contained a high quantity of bioactive compounds. Opuntia cladode contained high amounts of total phenols, total flavonoids, and total carotenoids and also exhibited strong antioxidant potential. Regarding prickly pear whole fruit, the aqueous extract exhibited the highest amounts of rutin (7.43 μg/g), quercitin (3.41 μg/g), gallic acid (84.74 μg/g), syringic acid (23.54 μg/g), whereas ethanolic extract showed the low quantity of rutin (0.40 μg/g), quercitin (0.19 μg/g), gallic acid (29.22 μg/g), and syringic acid (5.18 μg/g).
Moreover, prickly pear possesses effective antibacterial bioactive constituents against multi-drug-resistant bacteria and can be used for the prevention of different infectious diseases. The correlation analysis demonstrated phenolic contents, flavonoid contents, and carotenoids exhibited a substantial positive relationship with DPPH in an increasing manner. In this context, cladode is a valuable source of health-promoting compounds, fulfilling concurrently the promising antioxidant activity that can be utilized virtually as food complements, to tardiness lipid oxidization and healing from particular ailments via its free radicals scavenging ability. It would be interesting to conduct more research to inspect the role of bioactive components which responsible for these activities. Hence, more studies are necessary to estimate the antioxidant and antimicrobial efficiencies of their individual purified fractions. Furthermore, it was revealed in the current study that the concentration of phenolic compounds and antioxidant activity in prickly pear extracts is sufficient to be considered a possible source of antioxidant supplements. In general, it may have the potential to contribute to better bioactive compounds contents in the diet of children, mothers, and adolescents to combat mineral deficiency problems and prevent many diseases including osteoporosis and cardiovascular disorders. The demand for fortified foods in the market is huge. Hence, prickly pear extracts fortification in foodstuffs is a convenient way to eradicate malnutrition. This abundant tree in Pakistan can become a great source of income for the nation if this potential for highly nutritional food is exploited by industries and researchers.

FU N D I N G I N FO R M ATI O N
There was no funding received for this study from any funding organization.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon reasonable request.